Isolation and amplification of nucleic acid materials

ABSTRACT

The invention relates to methods for easily separating single stranded nucleic acid material from double stranded nucleic acid material in a sample containing both. By the right choice of at lease one chaotropic agent, preferably a guanidine salt, at a selected concentration and other suitable conditions such a chelating agents, pH and the like, it is possible to bind double stranded material to a solid phase such as silica particles, whereas single stranded material will not bind under those circumstances. By separating the silica particles from the sample the double stranded nucleic acid material is removed. It can easily be eluted from the silica particles. In a second step the single stranded material may be bound to a solid phase by selecting a different set of conditions. The particles can again be separated from the sample and the single stranded material may now be eluted. For very efficent separations, the process may be repeated. Following the separation of the two kinds of nucleic acid, either kind may be amplified. Methods of amplification are provided which do not need sequence data of the material to be amplified. In these methods a primer will be provided with an amplification motif and a random hybridization motif.

[0001] The invention relates to the field of purification andamplification of nucleic acids from nucleic acid containing startingmaterials, especially from biological materials such as urine, faeces,sperm, saliva, whole blood, serum or other body fluids, fractions ofsuch fluids such as leucocyte fractions (buffy coats), cell cultures andthe like, but also samples from the environment such as soil, water andthe like.

[0002] Until recently isolation and/or purification of nucleic acidsfrom complex mixtures as described above was a laborious, multi-stepprocedure. In EP 0389063, incorporated herein by reference, a simple andrapid purification of nucleic acid material from a complex mixture isdisclosed. This procedure comprises treating the complex mixture, suchas whole blood with a chaotropic agent in the presence of a nucleic acidbinding silica solid phase material under conditions that allow forbinding of all nucleic acid material to said solid phase and separatingsaid solid phase from the mixture. The reference shows that both singlestranded and double stranded nucleic acids are bound to the solid phaseif present in a mixture. The reference also discloses amplification(PCR) of a certain nucleic acid with a known sequence, suspected to bepresent in a mixture.

[0003] Thus said reference teaches a simple and rapid detection methodfor known nucleic acids suspected to be present in a sample.

[0004] In many cases the nature of the target nucleic acid (doubleatranded or single stranded) may not be known beforehand, or there maybe many different targets necessary to be analyzed. In these cases therapid but rather crude method described above may not be sophisticatedenough and further separations of the crude material may be wanted.Fractionation of mixtures of double- (ds) and single-stranded (ss)nucleic acids (NA) into single- and double-stranded forms is frequentlyneeded e.g. in the separation of labelled ss-NA probes from ds-hybrids,in the separation of in vitro transcripts from ds-DNA templates, and inthe separation of genomic DNA from mRNA. Currently, the separation ofdifferent kinds of nucleic acids can be accomplished by severaltechniques. Electrophoresis can be used to fractionate different formsof nucleic acids, because of differences in size and shape (1-3).Centrifugation takes advantage of differences in density (4), and morerecently the technology of high-performence liquid chromatography (HPLC)has been applied to separate and purify single- and double-stranded DNAand RNA molecules (5-8).

[0005] RNA purified from eukaryotic cells by the currently most widelyused procedure (9) appears to contain significant amounts of genomicDNA, an adaptation which reduces genomic DNA contamination of the ss-RNAfraction has recently been described (10).

[0006] It is not possible to look at single stranded and/or doublestranded material separately using the method of EP 0389063 because themethod does not discriminate between the two.

[0007] The present invention therefor provides a method for separatingsingle stranded nucleic acid material from double stranded nucleic acidmaterial comprising contacting a mixture of the both with a liquidcomprising a chaotropic agent and a nucleic acid binding solid phase,whereby the liquid has a composition such that double stranded nucleicacid binds to the solid phase and a substantial amount of singlestranded nucleic acid does not and separating the solid phase from theliquid. Suitable circumstances to arrive at such a separation can bedetermined by the person skilled in the art.

[0008] Circumstances under which double stranded material binds to thesolid material and single stranded material will vary, however importantparameters to obtain such differential binding are the concentration ofthe chaotropic agent, which should roughly be between 1-10 M, preferablybetween 3-6 M and particularly about 5 M; the concentration of chelatingagent, which in the case that EDTA is applied should be equal to orgreater than 10 mM and preferably not higher than 1 M; the pH of theaqueous solution in which the separation is carried out should be above2 when a thiocyanate is used as chaotropic agent and it should be below10 because otherwise there is a risk that the ds material will becomess. The temperature at which the process is carried out seems to benon-critical, however, it is probably best to keep it between 4° C. and60° C. An important aspect of the process is of course that the dsmaterial remains double stranded during the separation. Under thecircumstances as disclosed above this will normally be the case if theds nucleic acid is at least 50 bp long at 40% GC basepairs. The skilledartisan knows how this length may vary with lower or higher GC content.In Van Ness et al (26) and/or Thompson et al (27) it is shown that thewhole process depends on intricate interactions between a.o. the factorsmentioned above. Using this disclosure and the cited references theskilled artisan will be able to adjust the circumstances to his or herparticular process.

[0009] Chaotropic agents are a very important feature of the presentinvention. They are defined as any substance that can alter thesecondary, tertiary and/or quaternary structure of nucleic acids. Theyshould have no substantial effect on the primary structure of thenucleic acid. If nucleic acids are present associated with othermolecules, such as proteins, these associations can also be altered bythe same or different chaotropic agents. Many chaotropic agents aresuitable for use in the present invention, such as sodium iodide,potassium iodide, sodium (iso)thiocyanate, urea or guanidinium salts, orcombinations thereof. A preferred class of chaotropic agents accordingto the invention are guanidinium salts, of which guanidinium thiocyanateis most preferred.

[0010] By serendipity we found that ss-nucleic acid did not bind tosilica particles or diatomeous earth in the presence of buffer L11 (seeexamples), whereas ds nucleic acid did. Experiments with differentcircumstances showed that addition of Mg²⁺ or other positive (bivalent)ions to the unbound fraction was of great importance. The best resultswere obtained with a concentration of bivalent ion (Mg²⁺) about equal tothe concentration of the chelating agent (EDTA).

[0011] The solid phase to be used is less critical. Important is that itshould bind nucleic acids reversibly.

[0012] Many such materials are known, of which a number are siliciumbased, such as aluminium silicate and the like, preferably silica.Silica is meant to include SiO₂ crystals and other forms of siliconoxide, such as diatom skeletons, glass powder and/or particles andamorphous silicon oxide. The solid phase may be present in any form, itmay even be the vessel which contains the nucleic acid mixtures or apart of such a vessel. It may also be a filter or any other suitablestructure. Apart from silicium based materials other materials will alsobe suitable, such as nitrocellulose (filters), latex particles and otherpolymeric substances. A preferred form of the solid phase is aparticulate form, which allows for easy separation of bound and freematerial, for instance by centrifugation. The particle size of the solidphase is not critical. Suitable average particle sizes range from about0.05 to 500 μm. Preferably the range is chosen such that at least 80,preferably 90% of the particles have a size between the values justmentioned. The same holds true for the preferred ranges of which theaverage particle sizes are between 0.1 and 200 μm, preferably between 1and 200 μm. The binding capacity of a given weight of the particlesincreases with decreasing size, however the lower limit of the size iswhen particles cannot easily be redispersed after separation through forinstance centrifugation. This will be the case in starting material richin nucleic acids containing many nucleic acids of a higher molecularweight. The particles and the nucleic acids may form aggregates in thesecases. The person skilled in the art will be able to choose the rightparticle size for the particular application envisioned. The formationof aggregates may be avoided by using fractionated silica ordiatomaceous earth in a number of applications.

[0013] A further embodiment of the present invention is a method forisolating single stranded nucleic acid material from a mixture ofnucleic acid material, comprising the steps of subjecting the mixture toa method as described hereinabove and treating the supernatantcontaining the single stranded nucleic acid material with a secondliquid comprising a chaotropic agent and a second nucleic acid bindingsolid phase, whereby the second liquid has a compositon such that theresulting mixture of supernatant and second liquid allow for binding ofthe single stranded nucleic acid material to the second solid phase.

[0014] This way the double stranded nucleic acid material is removedfrom the crude mixture and the single stranded nucleic acid is purifiedfrom the remaining still crude mixture in another single step. Both thedouble stranded material and the single stranded material are reversiblybound to the respective solid phases, so that they may be easily elutedfrom said solid phases to undergo further analysis or other treatments.A very useful further treatment is the amplification of the (double orsingle stranded) nucleic acid material.

[0015] Both types can be amplified, or both types may be converted intoone another so that they can be amplified. The present inventionprovides in yet another embodiment a method for amplifying singlestranded nucleic acid material comprising the steps of hybridizing thesingle stranded nucleic acid with primers and elongating the probesusing an enzyme which adds nucleotides to the primer sequence using thehybridized single strand material as a template, whereby at least oneprimer comprises a random hybridizing sequence and an amplificationmotif.

[0016] Single-stranded nucleic acids purified in accordance with theinvention were used as input for a cDNA synthesis reaction using primerswith random 3′ ends (tagging primers) for the first and second strandsynthesis (see the outline in FIG. 7).

[0017] These tagged cDNAs are then amplified by using only one PCRprimer homologous to the PCR motif present in both tagging primers. Thetagging primer used in the first strand synthesis (TAG 20) has beenespecially designed to facilitate subsequent direct sequencing of theresultant PCR products.

[0018] In contrast with most other protocols (16-22) the describedmethod does not need any sequence data at all, and the majority ofamplified products can be visualized on ethidiumbromide stained agarosegels as discrete bands, which makes isolation and direct sequencing ofthe amplified cDNA feasible. The criteria for amplification are wellknown in the art. The length of suitable primers, suitable buffers,suitable melting temperatures for separating strands, suitablehybridization conditions can all be determined using standard handbooksin the field.

[0019] Of course the sequences which are exemplified can be variedwithout departing from the present invention. It is not so muchimportant what sequence is used as an amplification motif, as long as itis suitable for hybridization and primer extension purposes. Suitablelimits depend on the conditions which can be varied by the personskilled in the art. Usually primers will be at least 10 bases long andnot much longer than 100 bases.

[0020] The amplification embodiments of the invention are exemplifiedusing PCR (polymerase chain reaction). Other amplification methods areof course equally suitable.

[0021] The exemplified label (or tag) on the primers is DIG(digoxygenin). However other labels are available and well known in theart.

[0022] The invention will now be explained in further detail in thefollowing detailed description.

[0023] Separation/Isolation

MATERIALS AND METHODS

[0024] Source of nucleic acids.

[0025] Phage MS-2 ss-RNA (3569 nt), E. coli rRNA (16 and 23S; 1,7 kb and3,5 kb respectively), phage M13 ss-DNA (7599 nt) and HindIII digestedphage lambda ds-DNA were purchased from Boehringer (Mannheim, Germany).Rotavirus ds-RNA was purified from feces of an infected individual byprotocol Y/SC (11). Plasmid DNA was purified from E. coli HB101 asdescribed by Ish-Horowicz and Burke (13) followed by columnchromatography with Sepharose CL2B (Pharmacia, Inc. Uppsala, Sweden).Total NA was purified from E. coli by protocol Y/D (11).

[0026] Chemicals.

[0027] Guanidiniumthiocyanate (GuSCN) was obtained from Fluka (Buchs,Switzerland).

[0028] EDTA (Titriplex) and MgCl2.6H20 were obtained from Merck(Darmstadt, Germany). TRIS was obtained from Boehringer (Mannheim,Germany). The preparation of size-fractionated silica particles (silicacoarse, SC) and diatom suspension has been described (11). Triton X-100was from Packard (Packard Instrument Co., Inc., Downers Grove, Ill.).

[0029] Composition of buffers.

[0030] The lysis/binding buffer L6, washing buffer L2, and TE (10 mMTris.HCI, 1 mM EDTA; pH=8.0) have been described (11). 0.2M EDTA (pH8.0) was made by dissolving 37.2 g EDTA (Merck, Germany) and 4.4 g NaOH(Merck, Germany) in aqua in a total volume of 500 ml. Lysis/bindingbuffer L11 was made by dissolving 120 g of GuSCN in 100 ml 0.2M EDTA(pH=8.0) Binding buffer L10 was prepared by dissolving 120 g GuSCN in100 ml 0.35M TRIS.HCl (pH 6.4); subsequently 22 ml 0.2M EDTA (pH 8.0)and 9.1 g Triton X-100 were added and the solution was homogenized;finally 11 g of solid MgCl₂.6H₂O was added. The final concentration ofMgCl₂ in L10 is about 0.25M. L10 is stable for at least 1 month whenstored at ambient temperature in the dark.

[0031] Fractionation of ds-NA and ss-NA by protocol R.

[0032] The procedure is outlined in FIG. 1. A 50 μl specimen (containinga mixture of NA-types in TE buffer) was added to a mixture of 900 μl L11and 40 μl SC in an Eppendorf tube and was subsequently homogenized byvortexing. After 10 min. binding at room temperature, the tube wascentrifuged (2 min. at approx. 10.000×g) which resulted in asilica/ds-NA pellet (“initial silica pellet”) and a supernatantcontaining ss-NA.

[0033] To recover ss-NA forms (protocol R-sup), 900 μl of thesupernatant were added to a mixture of 400 μl L10 and 40 μl SC and ss-NAwas bound during a 10 min. incubation at room temperature. The tube wassubsequently centrifuged (15 sec. at approx. 10.000×g), and thesupernatant was discarded (by suction). The resulting pellet wassubsequently washed twice with 1 ml of L2, twice with 1 ml ethanol 70%(vol/vol) and once with 1 ml acetone. The silica pellet was dried (10min. at 56° C. with open lid in an Eppendorf heating block) and elutedin 50 μl TE buffer (10 min. at 56° C.; closed lid). After centrifugation(2 min. at approx. 10.000×g) the supernatant contains the ss-NA fraction

[0034] To recover ds-NA forms (protocol R-pellet) from the initialsilica-pellet, the remaining supernatant was discarded, and the silicapellet was washed twice with L11 to remove unbound ss-NA. The resultingsilica pellet was subsequently washed twice with L2, twice with ethanol70%, once with acetone, dried and eluted as described above. Aftercentrifugation (2 min. at approx. 10.000×g) the supernatant contains theds-NA fraction.

[0035] In the complete procedure (which takes about one hour) forfractionation of NA by protocol R, only two Eppendorf tubes are used.

[0036] Fractionation of genomic DNA and ss-NA.

[0037] Due to trapping of ss-NA into high-molecular-weight genomic DNA,protocol R as described above gives only low yields of ss-NA. This canbe circumvented by first isolating total NA by protocol Y/D (11), whichcauses some shearing of the high-molecular-weight genomic DNA,sufficient enough to prevent trapping of the ss-NA. Total NA thuspurified can subsequently be used as input for protocol R.

[0038] Gel electrophoresis.

[0039] In all experiments, NA was electrophoresed (8 to 10 V/cm) throughneutral agarose slab gels containing ethidiumbromide (1 μg/ml) in thebuffer system (40 mM TRIS-20 mM sodium acetate-2 mM EDTA adjusted to pH7.7 with acetic acid; ethidium bromide was added to a concentration of 1μg/ml of buffer) described by Aaij and Borst (14).

[0040] Hybridization.

[0041] DNA fragments were transferred to nitrocellulose filters by theprocedure of Southern (15) and hybridized with [alpha-³²P]dCTP labelledpHC624 (16) prepared by random labeling (Boehringer, Germany).Hybridization conditions were as described previously (12).

RESULTS

[0042] Comparison of different GuSCN-containing lysisbuffers withrespect to the binding of different NA-types to silica particlesrevealed that only doublestranded forms were bound when using L11 (whichis about 100 mM for EDTA) as binding buffer; on the other hand bothdouble- and single-stranded forms were bound in binding buffer L6 (whichis about 20 mM for EDTA) (Table 1). These observations formed the basisfor the development of a protocol (Protocol R) for the fractionation ofsingle-stranded nucleic acids and double-stranded nucleic acids (FIG. 1)

[0043] Once double-stranded nucleic acid is bound by silica particles inL11, a brief centrifugation will separate the silica/ds-NA pellet fromthe supernatant containing the single-stranded forms. Addition of thissupernatant to a mixture of silica particles and binding buffer L10(which is about 250 mM for Mg²⁺) the binding of single-stranded nucleicacids to the silica particles is restored. Double-stranded andsingle-stranded forms can subsequently be purified by washing andeluting the silica-NA complexes (protocol R). Double-stranded nucleicacid is recovered from the initial silica-pellet (protocol R-pellet),whereas single-stranded forms are recovered from the initial supernatant(protocol R-sup).

[0044] For optimization of protocol R we performed reconstructionexperiments in which previously purified or commercially available,nucleic acids were mixed and subsequently fractionated by protocol R.

[0045] Fractionation of a mixture of double-stranded DNA andsingle-stranded DNA.

[0046] The fractionation of a ds-DNA/ss-DNA mixture, into doublestranded- and single stranded forms is shown in FIG. 2. The recoveryestimated from the band intensity of the ethidium bromide stained gelfor ss-DNA was about 50%, the estimated recovery of ds-DNA in the rangeof 500 bp to 4,6 kb was 80%-90% [similar recoveries were obtained fords-DNA fragments in the range of 100-500 bp (not shown)], largerfragments were significantly sheared as noted before (11). At the levelof detection by UV-illumination, fractionation into ds- and ss-forms wascomplete.

[0047] Fractionation of a mixture of double-stranded RNA andsingle-stranded RNA.

[0048]FIG. 3 shows the fractionation of a mixture of ds-RNA (humanRotavirus genome segments 1-11; for review see 14) and ss-RNA (phage MS2RNA) into double stranded- and single stranded forms. The estimatedrecovery of ds-RNA and ss-RNA was at least 80%. At the level ofdetection by UV-illumination, fractionation into ds- and ss-forms wascomplete.

[0049] Fractionation of a mixture of double-stranded DNA andsingle-stranded RNA.

[0050] In FIG. 4 it is shown that ds-DNA can also efficiently beseparated from ss-RNA.

[0051] Again are the recoveries for both fractions at least 80%. Similarresults were obtained when E. coli rRNA (23S and 16S) was used as ss-RNAinput (not shown).

[0052] In the experiments described above, fractionation of ds- andss-NA forms (as judged by visual inspection of band intensities afterethidiumbromide staining and UV illumination) appeared to be complete.In order to establish the performance of the fractionation procedure fora mixture of ds-DNA and ss-RNA into ss- and ds-forms, NA purified byprotocol R-sup from such a mixture was studied by Southern blotting andhybridization with a ³²P-labelled DNA probe, homologous to the ds-DNAused as input for fractionation. This experiment revealed that the ss-NAfraction contained less than 0,1% of the ds-DNA input (FIG. 5).

[0053] Fractionation of a mixture of genomic DNA and single-strandedRNA.

[0054] When we investigated the separation of high-molecular-weight(genomic) dsDNA and ss-RNA by direct fractionation using E. coli asinput for protocol R, it appeared that the ds-DNA fraction was heavilycontaminated with rRNA (FIG. 6, lanes 6 and 7), and ss-RNA recovery waslow (FIG. 6, lanes B and 9). This was likely due to trapping of RNA intohigh-molecular-weight (genomic) ds-DNA when silica/NA complexes wereformed. On the other hand no genomic DNA was observed in the ss-RNAfraction. Total nucleic acid, which was first isolated using thestandard protocol Y/D (11), and hereafter used as input material inprotocol R showed significantly higher recoveries of the ss-RNA fraction(FIG. 6, lanes 2 and 5).

[0055] Amplifications

MATERIALS AND METHODS

[0056] Source of nucleic acids.

[0057] HIV-1 RNA was isolated from a virus culture (23), phage MS-2 RNAwas purchased from Boehringer (Mannheim, Germany) and the 7.5 KbPoly(A)Tailed RNA and the 100 bp ladder used as a marker were purchasedfrom Life Technologies (Gaithersburg, Md., USA). The PCR TA3 cloningvector was obtained from Promega (Madison, USA). Plasmids 5′ NOT Hxb₂ENN(24) {containing the GAG and POL genes of HIV-1 from nucleotide638-4647} and 168.1 RTN (24) {containing the ENV gene of HIV-1 fromnucleotide 5674-8474} were purified as described by Ish-Horowicz andBurke (13) followed by protocol R-pellet as described in the examples.The plasmid pHCrec used as a positive control in the PCR experiments wasmade by a low annealing PCR on lambda DNA (Boehringer) using PCR primerRB 8 (see below). The discrete PCR products were purified using protocolY/D (11) and subsequently cloned in a PCR III vector (Invitrogen) . Therevealing plasmid, pHCrec with a approximately 600 bp insert wassubsequently purified from E. coli HB101 as described by Ish-Horowiczand Burke (13) followed by column chromatography with Sepharose CL2B(Pharmacia, Inc. Uppsala, Sweden).

[0058] Chemicals and enzymes

[0059] EDTA, KCl, MgCl₂.6H₂O, NaCl and tri-Sodium citrate dihydrate wereobtained from Merck (Darmstadt, Germany). TRIS and BSA were obtainedfrom Boehringer (Mannheim, Germany). Triton X-100 was obtained fromPackard (Packard Instruments Co., Inc., Downers, Ill., USA). SodiumDodecylsulfate (SDS) was obtained from Serva (Heidelberg, Germany).

[0060] The dNTP's and Dextran Sulphate were obtained from Pharmacia(Uppsala, Sweden).

[0061] The chemicals used in protocol R have been described herein.

[0062] Reverse transcriptase SuperScript II was purchased from LifeTechnologies (Gaithersburg, Maryland, USA). DNA polymerase Sequenase 2was obtained from Amersham (United Kingdom). Ampli-Taq DNA polymerasewas obtained from Perkin Elmer (Norwalk, USA). RNAse H was obtained fromBoehringer (Mannheim, Germany). Salmon sperm DNA was obtained from Sigma(St. Louis, USA).

[0063] Composition of buffers and solutions.

[0064] The preparation of the buffers used in protocol R have beendescribed herein, except that the lysis buffer and washing buffers (L10,L11, and L2) used in protocol R for the isolation of nucleic acids werefiltered through a column packed with Diatoms (11) in order to removeany endogenous nucleic acids in the lysis buffer and washing buffers.

[0065] The 10 x reverse transcription buffer (CMB1) consists of 100 mMTris.HCl (pH 8.5), 500 mM KCl and 1% Triton X-100.

[0066] The 10 x PCR buffer consists of 500 mM Tris.HCl (pH 8.3), 200 mMKCl and 1 mg/ml BSA.

[0067] The elution buffer Tris/EDTA (TE, pH 8.0) consists of 10 mMTris.HCl (pH 8.0) and 1 mM EDTA (pH 8.0).

[0068] Oligonucleotides.

[0069] The first strand primer TAG 20:

[0070]5′ GACAGAATGCCGAAATGACCCCNNNNNG3′

[0071] The second strand primer TAG 7:

[0072] 5′DIG-GACAGAATGCCGAAATGANNNNNG3′

[0073] The PCR primer RB 8:

[0074] 5′GACAGAATGCCGAAATGA3′

[0075] underlined: PCR motif

[0076] bold: motif for direct sequencing

[0077] N=A, T, C, or G

[0078] Protocol for first strand synthesis.

[0079] ss-RNA, present in the commercially available reversetranscriptases, appeared to produce unwanted side products when used infirst strand synthesis. To overcome this problem reverse transcriptasewas first pretreated in a mixture for cDNA synthesis lacking exogenouslyadded primers:

[0080] 1 μl SuperScript II (200 U/μl)

[0081] 1 μl CMB1 (10 x)

[0082] 0.5 μL MgCl₂ (100 mM)

[0083] 0.4 μL dNTP's (25 μM each)

[0084] 7.1 μL H₂O

[0085] Incubate 15 min. at 37° C.

[0086] Nucleic acids (20 μl) purified by protocol R-sup were incubatedfor 5 min. at 60° C. and thereafter quenched on ice. Subsequently thefollowing mixture was added:

[0087] 3 μl CMB1 (10 x)

[0088] 1 μl TAG 20 (100 ng/μl)

[0089] 1.5 μL MgCl₂ (100 mM)

[0090] 1.2 μl dNTP's (25 mM each)

[0091] 3.3 μl H₂O

[0092] Finally 10 μl of the preincubated Superscript II (SS II) wasadded and the resulting mixture was incubated for 30 min. at 42° C.

[0093] After the reverse transcription reaction SS II was inactivated byincubating the mixture for 5 min. at 80° C., and the mixture wassubsequently cooled down to room temperature. In order to convert theRNA/DNA hybrids into single-stranded cDNA twenty units of RNAse H wereadded to the mixture and incubated for 60 min. at 37° C. Thesingle-stranded cDNA was subsequently isolated using protocol R-sup. Thesingle-stranded cDNA was eluted in 40 μl TE and 20 μl was used as inputfor second strand synthesis.

[0094] Protocol for second strand synthesis.

[0095] To twenty microliter of single-stranded cDNA, the followingmixture was added (on ice):

[0096] 4 μl CMB1 (10 x)

[0097] 1 μl TAG 7-DIG* (100 ng/μl)

[0098] 2 μl MgCl₂ (100 mM)

[0099] 1.6 μl dNTP's (25 mM each)

[0100] 0.2 μl Sequenase 2 (13 U/μl)

[0101] 11.2 μl H₂O

[0102] The mixture was incubated for 10 min. on ice, and subsequentlyfor 60 min. at 37° C. After the second strand synthesis thedouble-stranded cDNA was isolated using protocol R-pellet. Thedouble-stranded cDNA was eluted in 40 μl TE. Twently microliter wastaken of and 2 μl was used as input for PCR. The remaining 18 μl wasstored at −20° C.

[0103] Protocol for the polymerase chain reaction.

[0104] Two microliters of double-stranded cDNA was added to 48 μl of aPCR mixture consisting of:

[0105] 18 μl TE (pH 8.0)

[0106] 1 μl RB 8 (100 ng/μl)

[0107] 5 μl PCR buffer (10 x)

[0108] 0.9 μl MgCl₂ (100 mM)

[0109] 0.2 μl dNTP's (100 μM)

[0110] 0.1 μl dUTP* (25 μM)

[0111] 0.3 μl Ampli Taq (5 U/μl)

[0112] 22.5 μl H₂O

[0113] After incubation for 5 min. at 95° C. the sample was subjected to45 cycles of amplification in a DNA thermal cycler (type 480; PerkinElmer Cetus). A cycle consisted of denaturation for 1 min. at 95° C.,annealing for 1 min. at 63° C., and elongation for 2 min. at 72° C.After cycling the sample was incubated for 8 min. at 72° C., andsubsequently the temperature was lowered to 4° C. Twentyfive microliterof the PCR product was examined by agarose gel electrophoresis andethidiumbromide staining. In every experiment TE was used as a negativeextraction control and as a negative PCR control.

[0114] Gel electrophoresis.

[0115] In all experiments, the nucleic acids were electrophoresed (8 to10 V/cm) through neutral agarose slab gels containing ethidiumbromide (1μg/ml) in the buffer system as described by Aaij and Borst (14)

[0116] Hybridization.

[0117] DNA fragments were detected after Southern blotting (15) byhybridization with ³²P-labelled probes representing the entire GAG, POL,and ENV genes of HIV-1 [plasmid 5′ NOT Hxb₂ENN and plasmid 168 1 RTN](10).

RESULTS

[0118] In parallel, 10⁵ molecules of HIV-1 RNA (23) and negativecontrols (TE) were extracted using protocol R-sup. The resultingsingle-stranded nucleic acids were amplified by the non-selective RT-PCRas disclosed above, resulting in a discrete banding pattern for HIV-1RNA, and no amplification products in the TE controls (FIG. 8). Thevariation between the duplicates is a reflection of the non-selectivityof the procedure. As a control for the efficiency of the PCR part of theprocedure we used an input of 0, 6, 60 and 600 molecules of the plasmidpHCrec.

[0119] In order to confirm the HIV-origin of the bands visible in FIG.8A, we performed a Southern blot hybridization under high stringencyconditions with ³²P-labelled probes encompassing almost the entire HIV-1genome (FIG. 8B). This experiment showed that most of the bands visibleby UV-illumination hybridized with the HIV-1 probe. The bands that didnot hybridize with the probe might be homologous to parts of the HIV-1genome other than those present in the probe or might originate fromsingle-stranded RNA present in the HIV-1 RNA preparation (e.g. cellularmRNA) or ss-RNA present in Superscript II, which was not converted tods-hybrids during the preincubation of the SuperScript II.

[0120] Similar results were obtained with other single-stranded RNAssuch as hepatitis C virus RNA, phage MS2 RNA, and the 7.5 KbPoly(A)-Tailed RNA (results not shown).

[0121] It is concluded that the described procedure can be used toamplify single stranded RNA targets (present in e.g. serum) to a seriesof discrete bands in agarose gels. The discrete bands can be purifiedfrom agarose gels, cloned in e.g. a bacterial vector and the clones cansubsequently be sequenced. Due to the fact that one of the taggingprimers (TAG 20) harbours a sequence motif it is possible to sequencethe discrete bands without cloning, after the bands are purified fromgel. The method described here is useful in isolating and characterizingunknown sequences present in clinical samples (e.g. viral sequences) orfor the amplification of cDNAs from transcripts without having anysequence data.

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[0149] Binding of different NA-types to silica particles in differentlysisbuffers: similar results were obtained using diatoms rather thansilica particles (data not shown).

1. A method for separating single stranded nucleic acid material fromdouble stranded nucleic acid material comprising contacting a mixture ofthe both with a liquid comprising a chaotropic agent and a nucleic acidbinding solid phase, whereby the liquid has a composition such thatdouble stranded nucleic acid binds to the solid phase and a substantialamount of single stranded nucleic acid does not and separating the solidphase from the liquid.
 2. A method according to claim 1 whereby theliquid comprises at least 100 mM EDTA and comprising a guanidinium salt,preferably guanidinium thiocyanate as a chaotropic agent.
 3. A methodaccording to claim 1 or 2 , whereby the solid phase is silicium based.4. A method according to claim 3 whereby the solid phase is silica.
 5. Amethod according to claim 4 whereby the silica is in the form ofparticles having a size between 0.05 and 500, preferably 0.1 and 200 μm.6. A method according to anyone of the aforegoing claims whereby thesolid phase is separated from the supernatant by centrifugation.
 7. Amethod for isolating single stranded nucleic acid material from amixture of nucleic acid material, comprising the steps of subjecting themixture to a method according to anyone of the aforegoing claims andtreating the supernatant containing the single stranded nucleic acidmaterial with a second liquid comprising a chaotropic agent and a secondnucleic acid binding solid phase, whereby the second liquid has acompositon such that the resulting mixture of supernatant and secondliquid allow for binding of the single stranded nucleic acid material tothe second solid phase.
 8. A method for amplifying single strandednucleic acid material comprising the steps of hybridizing the singlestranded nucleic acid with primers and elongating the probes using anenzyme which adds nucleotides to the primer sequence using thehybridized single strand material as a template, whereby at least oneprimer comprises a random hybridizing sequence and an amplificationmotif.
 9. A method according to claim 8 whereby at least one primercomprising a random hybridization sequence and an amplification sequencefurther comprises a label.
 10. A method according to claim 8 or 9whereby at least one primer comprising a random hybridizing sequence andan amplification motif further comprises a direct sequencing motif. 11.A method for isolating and amplifying single stranded nucleic acidmaterial originally present in a mixture of nucleic acids comprisingsubjecting the mixture to a method according to anyone of claims 1-7followed by subjection of the isolated material to a method according toanyone of claims 8-10.
 12. A method according to any one of theaforegoing claims whereby the single stranded nuclec acid materialcomprises mRNA.
 13. A method according to claim 12 whereby the mRNA isconverted into cDNA.
 14. A method according to anyone of the aforegoingclaims comprising a gel electrophoresis step.
 15. A method according toanyone of the aforegoing claims followed by a sequencing step.